2-[[6-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]naphthalene-1,5-disulphonic acid, sodium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 19, 1992 to July 27, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

EEC Directive 84/449/EEC B.14. Other Effects –Mutagenicity Salmonella typhimurium Reverse Mutation Test

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals "Genetic Toxicology: Salmonella typhimurium, Reverse Mutation Assay" Adopted: 26 May 83, No. 471

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US EPA

Version / remarks:

New and Revised Health Effects Test Guidelines October 1984 (U.S.) Environmental Protection Agency Washington, DC (PB 84-233295).
HG - Gene Muta - s. typhimurium, October 1984

Principles of method if other than guideline:

Prival M.J. and V.D. Mitchell: Analysis of a method for testing azo dyes for mutagenic activity in Salmonella typhimurium in the presence of flavin mononucleotide and hamster liver S9. Mutation Res. 97, 103-116, 1982

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[[6-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]naphthalene-1,5-disulphonic acid, sodium salt

EC Number:

280-744-2

EC Name:

2-[[6-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]naphthalene-1,5-disulphonic acid, sodium salt

Cas Number:

83763-57-9

Molecular formula:

C24H11ClF2N5Na3O10S3 C24H14ClF2N5O10S3.xNa C24H13ClF2N5NaO10S3 [724.014 gr/mol]

IUPAC Name:

sodium 6-(2-{6-[(5-chloro-2,6-difluoropyrimidin-4-yl)amino]-1-hydroxy-3-sulfonaphthalen-2-yl}diazen-1-yl)-5-sulfonaphthalene-1-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

Reactive Orange 64

Specific details on test material used for the study:

No further details specified in the study report.

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

0, 8, 40, 200, 1000, 5000 μg/plate

The doses for the first trial were routinely determined on the basis of a standard protocol: if not limited by solubility 5000 ug or 5 ul per plate were used as the highest dose.

Vehicle / solvent:

The solvent employed for Congo red was deionized water, and for other positive controls DMSO. Levafix orange E-3GA was dissolved in deionized water.
The solvent used was chosen out of the following solvents, in the order given: water, methanol, ethanol, acetone, DMSO, DMF, and ethylene glycol dimethylether according to information given by the internal sponsor.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

congo red

other: Nitrofurantoin; 4-nitro-1,2-phenylene diamine; 2-aminoanthracene; benzidine

Details on test system and experimental conditions:

The initial plate incorporation test followed the directions of Ames et al. (1973a, 1975) and Maron and Ames (1983).
For the mutant count, four plates were used, both with and without 59 mix, for each strain and dose. An equal number of plates, filled with the solvent minus the test substance, comprised the negative control. Each positive control also contained four plate per strain. The amount of solvent for the test substance and for the controls was 0.1 ml/plate.
The doses for the first trial were routinely determined on the basis of a standard protocol: if not limited by solubility 5000 μg or 5 μl per plate were used as the highest dose. At least four additional doses were routinely used as progressive dilutions of the top dose. If less than three doses were used for assessment, at least two repeats were performed. The results of the first experiment were then considered as a pre-test for toxicity. However, in case of a positive response or if at least three doses could be used for assessment, the first trial was included in the assessment. If the second test confirmed the results of the first, no additional repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first experiment.
Because the first assessable trial showed no mutagenic effects of the test substance, the repeat was performed as pre-incubation test. Pre-incubation for 30 minutes at 37 °C was used.
The doses of this trial were determined on the basis of the results of the plate incorporation assay.
Due to the negative outcome of these two trials and due to the structure of the compound an additional repeat was performed according to Prival and Mitchell (1982) due to the chemical structure of the compound. Pre-incubation was performed in a water bath at 30 °C for 30 minutes. At the end of the pre-incubation period 2 ml of molten soft agar were added to the tubes, the content mixed and plated.
For the mutant count, four plates were used for each strain and dose. An equal number of plates, filled with the solvent minus the test substance, comprised the negative control.
Each positive control also contained four plates per strain.
In experiments without S9 mix buffer was used as replacement.
The doses of this trial were determined on the basis of the results of the pre-incubation assay.
The toxicity of the substance was assessed in three ways.
The first method was a gross appraisal of background growth on the plates for mutant determination. If a reduction in background growth was observed, it was indicated in the tables by the letter "b" after the mutant count. Where only a single "b", without any other values, is noted for a concentration, this "b" represents four plates with reduced background growth. (The same applies to the signs "c", "v", "p", “n" or "%", which may also be used in the tables.) Secondly, a toxic effect of the substance was assumed when there was a Thirdly, the titer was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix.
The bacterial suspensions were obtained from 17-hour cultures in nutrient broth, which had been incubated at 37 °C and 90 rpm. These suspensions were used for the determination of mutant counts. No standardized procedure was employed to set the bacterial suspensions at a defined density of viable cells per milliliter, since the chosen method of incubation normally produces the desired density. However the numbers of viable cells were established in a parallel procedure by determining the titers.
The dilution of bacterial suspensions used for the determination of titers was 1:1,000,000. Titers were determined under the same conditions as were the mutations, except that the histidine concentration in the soft agar was increased from 0.5 mM to 2.5 mM to permit the complete growth of bacteria.
The tests were performed both with and without S9 mix.
The count was made after the plates had been incubated for 48 hours at 37 °C. If no immediate count was possible, plates were temporarily stored in a refrigerator.

Rationale for test conditions:

The initial plate incorporation test followed the directions of Ames et al. (1973a, 1975) and Maron and Ames (1983).

Evaluation criteria:

The following criteria determined the acceptance of an assay:
a) The negative controls had to be within the expected range, as defined by published data (e.g. Maron and Ames, 1983) and/or the laboratories' own historical data.
b) The positive controls had to show sufficient effects, as defined by the laboratories' experience.
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.
An assay which did not comply with at least one of the above criteria was not used for assessment. Furthermore, the data generated in this assay needed to be confirmed by two additional independent experiments. Even if the criteria for points (a), (b) and (c) were not met, an assay was accepted if it showed mutagenic activity of the test compound.

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result.
For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.
In case of questionable results, investigations should continue, possibly with modification until a final evaluation is possible.

Statistics:

Not specified in the study report.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Plate incorporation assay
As may be seen, there was no indication of a bacteriotoxic effect of Levafix Orange E-3GA at doses of up to and including 5000 μg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well.
None of the four strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix.

Pre-incubation assay
As may be seen, there was no indication of a bacteriotoxic effect of Levafix Orange E-3GA at doses of up to and including 1000 μg per tube. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. 5000 μg per plate had only a weak, strain-specific bacteriotoxic effect, and could nevertheless be used for assessment purposes.
None of the four strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls and thus confirmed the results of the plate incorporation method.
The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

Assay according to Prival and Mitchell
As may be seen, there was no indication of a bacteriotoxic effect of Levafix Orange E-3GA at doses of up to and including 5000 μg per tube. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well.
None of the four strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls and thus confirmed the results of the plate incorporation and pre-incubation method.
The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, benzidine, Congo red and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

Any other information on results incl. tables

Summary of the Results With Levafix Orange E-3GA in the Salmonella Microsome Test Plate Incubation Method

S9 mix	TA 1535	TA 100	TA 1537	TA 98
Without	-ve	-ve	-ve	-ve
With	-ve	-ve	-ve	-ve

-ve = negative

 

Summary of the Results With Levafix Orange E-3GA in the Salmonella/Microsome Test Pre-incubation Method

S9 mix	TA 1535	TA 100	TA 1537	TA 98
Without	-ve	-ve	-ve	-ve
With	-ve	-ve	-ve	-ve

-ve = negative

 

Summary of the Results With Levafix Orange E-3GA in the Salmonella/Microsome Test Prival Assay

S9 mix	TA 1535	TA 100	TA 1537	TA 98
Without	-ve	-ve	-ve	-ve
With	-ve	-ve	-ve	-ve

-ve = negative

 

Summary of Mean Values Without S9 Mix

Group	Strain
	TA 1535	TA 100	TA 1537	TA 98
μg/plate 0 8 40 200 1000 5000 Na-azide NF 4-NPDA	17 16 17 17 10 15 296	66 59 70 78 55 67   203	7 8 7 7 4 4     52	18 16 18 17 13 19     67
μg/tube 0 8 40 200 1000 5000 Na-azide NF 4-NPDA	8 8 6 8 6 6 387	66 56 63 54 56 63   295	8 7 10 10 9 5     55	17 17 18 15 13 12     70
μg/tube 0 8 40 200 1000 5000 Na-azide NF 4-NPDA	7 7 7 10 7 6 121	78 84 80 71 84 75   277	8 4 4 7 6 5     43	22 12 11 16 16 14     44

 

Summary of Mean Values With S9 Mix

Group	Strain
	TA 1535	TA 100	TA 1537	TA 98
μg/plate 0 8 40 200 1000 5000 2-AA	18 18 22 20 18 14 98	79 79 98 91 72 83 539	11 11 9 7 7 5 69	22 28 24 23 19 20 442
μg/tube 0 8 40 200 1000 5000 2-AA	10 10 8 8 9 7 51	65 75 78 84 67 62 460	7 6 8 9 8 6 130	23 20 20 20 16 11 560
μg/tube 0 8 40 200 1000 5000 Benzidine Congo red 2-AA	14 14 9 10 10 9     90	155 159 170 170 157 157     1329	17 16 18 16 19 7     264	24 31 26 22 21 19 79 53

 

Summary of historical negative and positive controls of experiments performed from January to June 1991 using mean values presented as medians (Z) and semi-Q range (QR)

Compound and S9 Mix		Strain
		TA 1535		TA 100		TA 1537		TA 98
		Z	QR	Z	QR	Z	QR	Z	QR
Water DMSO DMF Methanol Ethanol Acetone EGDE^2	- - - - - - -	12 13 9 11 12 10 11	3 2 - 2 1 - 3	111 113 80 105 96 55 108	10 14 -- 14 15 -- 5	9 10 7 8 9 5 8	2 2 - 2 2 - 1	28 30 23 29 31 21 23	5 3 - 5 5 - 8
Na-azide NF 4-NPDA	- - -	623	102	398	56	49	10	89	20
30% Water DMSO DMF Methanol Ethanol Acetone EGDE^2	+ + + + + + +	16 18 11 23 19 14 15	3 3 - 5 3 - 4	152 154 84 152 127 84 132	15 11 -- 7 17 -- 6	12 12 9 10 10 14 8	2 2 - 3 3 - 1	38 40 29 48 43 18 40	7 7 - 10 6 - 9
2-AA	+	182	33	800	163	83	24	472	405
10% Water DMSO Methanol	+ + +	15 16 --	- 3 -	102 132 150	- 5 -	5 10 --	- 1 -	46 39 --	- 4 -
2-AA	+	208	48	1408	216	314	14	754	369

²) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from July to December 1991 using mean values presented as medians (Z) and semi-Q range (QR)

Compound and S9 Mix		Strain
		TA 1535		TA 100		TA 1537		TA 98
		Z	QR	Z	QR	Z	QR	Z	QR
Water Buffer DMSO DMF Methanol Ethanol Acetone EGDE^2	- - - - - - - -	12 13 12 7 10 12 12 14	3 2 3 - 1 4 2 3	89 97 92 75 84 80 87 107	10 10 15 - 11 8 6 22	9 8 9 7 8 8 8 8	3 1 1 - 1 3 1 1	27 25 24 17 25 23 26 26	4 2 4 - 3 4 4 5
Na-azide NF 4-NPDA	- - -	605	122	339	55	53	9	79	17
30% Water Buffer DMSO DMF Methanol Ethanol Acetone EGDE^2	+ + + + + + + +	19 17 19 11 25 18 18 22	4 - 3 - - 5 2 4	138 159 130 142 134 119 111 144	21 - 11 - - 19 9 11	13 13 10 9 12 11 13 13	2 - 2 - - 2 - 3	33 38 33 32 37 37 28 32	4 - 4 - - 2 11 3
2-AA	+	164	38	727	139	91	32	520	161
10% Water Buffer DMSO DMF Methanol Ethanol Acetone EGDE^2	+ + + + + + + +	16 14 16 15 16 19 17 20	4 - 2 - - 3 - 2	113 94 118 114 111 94 112 153	18 - 14 6 - 6 - 11	10 10 10 11 9 12 11 11	3 - 3 - - 2 - 1	33 34 31 21 29 32 32 34	5 - 3 - - 2 - 5
2-AA	+	197	50	1431	260	304	116	1097	207

²) Ethylene glycol dimethylether

Applicant's summary and conclusion

Conclusions:

Levafix Orange E-3GA is considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the Prival modification of the Salmonella/microsome test.
Therefore, the substance is not classified in accordance with CLP criteria.

Executive summary:

Levafix Orange E-3GA was initially investigated using the Salmonella/ microsome plate incorporation test for point mutagenic effects in doses of up to 5000 μg per plate of four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98.

 

In the plate incorporation assay doses of up to and including 5000 μg per plate did not cause any bacteriotoxic effects:

Total bacteria counts remained unchanged and no inhibition of growth was observed.

 

In the plate incorporation assay evidence of mutagenic activity of Levafix Orange E-3GA was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

 

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

 

In an independent repeat Levafix Orange E-3GA was investigated using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 μg per plate after pre-incubation for 30 minutes on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98.

 

Doses up to and including 1000 μg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At 5000 μg per plate, the substance had only a weak strain-specific baceriotoxic effect, so that this dose could nevertheless be used for assessment purposes.

 

Evidence of mutagenic activity of Levafix Orange E-3GA was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

 

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

 

Levafix Orange E-3GA was investigated in second independent repeat using the Salmonella/microsome test, modified with S9 mix according to Prival and Mitchell, for point mutagenic effects in doses of up to 5000 μg per tube on the same strains.

 

Doses of up to and including 5000 μg per tube did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed.

 

Evidence of mutagenic activity of Levafix Orange E-3GA was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

 

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, benzidine, Congo red and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls .

 

Therefore, Levafix Orange E-3GA was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the Prival modification of the Salmonella/microsome test.